Biochemical pathways to α-ketoglutarate, a multi-faceted metabolite.

α-Ketoglutarate (AKG) also known as 2-oxoglutarate is an essential metabolite in virtually all organisms as it participates in a variety of biological processes including anti-oxidative defence, energy production, signalling modules, and genetic modification. This keto-acid also possesses immense commercial value as it is utilized as a nutritional supplement, a therapeutic agent, and a precursor to a variety of value-added products such as ethylene and heterocyclic compounds. Hence, the generation of KG in a sustainable and environmentally-neutral manner is a major ongoing research endeavour. In this mini-review, the enzymatic systems and the metabolic networks mediating the synthesis of AKG will be described. The importance of such enzymes as isocitrate dehydrogenase (ICDH), glutamate dehydrogenase (GDH), succinate semialdehyde dehydrogenase (SSADH) and transaminases that directly contribute to the formation of KG will be emphasized. The efficacy of microbial systems in providing an effective platform to generate this moiety and the molecular strategies involving genetic manipulation, abiotic stress and nutrient supplementation that result in the optimal production of AKG will be evaluated. Microbial systems and their components acting via the metabolic networks and the resident enzymes are well poised to provide effective biotechnological tools that can supply renewable AKG globally.

Metabolic defence against oxidative stress: the road less travelled so far.

Bacteria have survived, and many have thrived, since antiquity in the presence of the highly-reactive chalcogen-oxygen (O2 ). They are known to evoke intricate strategies to defend themselves from the reactive by-products of oxygen-reactive oxygen species (ROS). Many of these detoxifying mechanisms have been extensively characterized; superoxide dismutase, catalases, alkyl hydroperoxide reductase and the glutathione (GSH)-cycling system are responsible for neutralizing specific ROS. Meanwhile, a pool of NADPH-the reductive engine of many ROS-combating enzymes-is maintained by metabolic enzymes including, but not exclusively, glucose-6 phosphate dehydrogenase (G6PDH) and NADP-dependent isocitrate dehydrogenase (ICDH-NADP). So, it is not surprising that evidence continues to emerge demonstrating the pivotal role metabolism plays in mitigating ROS toxicity. Stemming from its ability to concurrently decrease the production of the pro-oxidative metabolite, NADH, while augmenting the antioxidative metabolite, NADPH, metabolism is the fulcrum of cellular redox potential. In this review, we will discuss the mounting evidence positioning metabolism and metabolic shifts observed during oxidative stress, as critical strategies microbes utilize to thrive in environments that are rife with ROS. The contribution of ketoacids-moieties capable of non-enzymatic decarboxylation in the presence of oxidants-as ROS scavengers will be elaborated alongside the metabolic pathways responsible for their homeostases. Further, the signalling role of the carboxylic acids generated following the ketoacid-mediated detoxification of the ROS will be commented on within the context of oxidative stress.

Phospho-transfer networks and ATP homeostasis in response to an ineffective electron transport chain in Pseudomonas fluorescens.

Although oxidative stress is known to impede the tricarboxylic acid (TCA) cycle and oxidative phosphorylation, the nutritionally-versatile microbe, Pseudomonas fluorescens has been shown to proliferate in the presence of hydrogen peroxide (H2O2) and nitrosative stress. In this study we demonstrate the phospho-transfer system that enables this organism to generate ATP was similar irrespective of the carbon source utilized. Despite the diminished activities of enzymes involved in the TCA cycle and in the electron transport chain (ETC), the ATP levels did not appear to be significantly affected in the stressed cells. Phospho-transfer networks mediated by acetate kinase (ACK), adenylate kinase (AK), and nucleoside diphosphate kinase (NDPK) are involved in maintaining ATP homeostasis in the oxidatively-challenged cells. This phospho-relay machinery orchestrated by substrate-level phosphorylation is aided by the up-regulation in the activities of such enzymes like phosphoenolpyruvate carboxylase (PEPC), pyruvate orthophosphate dikinase (PPDK), and phosphoenolpyruvate synthase (PEPS). The enhanced production of phosphoenolpyruvate (PEP) and pyruvate further fuel the synthesis of ATP. Taken together, this metabolic reconfiguration enables the organism to fulfill its ATP need in an O2-independent manner by utilizing an intricate phospho-wire module aimed at maximizing the energy potential of PEP with the participation of AMP.

Zinc toxicity and ATP production in Pseudomonas fluorescens.

AIMS: To identify the molecular networks in Pseudomonas fluorescens that convey resistance to toxic concentrations of Zn, a common pollutant and hazard to biological systems. METHODS AND RESULTS: Pseudomonas fluorescens strain ATCC 13525 was cultured in growth medium with millimolar concentrations of Zn. Enzymatic activities and metabolite levels were monitored with the aid of in-gel activity assays and high-performance liquid chromatography, respectively. As oxidative phosphorylation was rendered ineffective, the assimilation of citric acid mediated sequentially by citrate lyase (CL), phosphoenolpyruvate carboxylase (PEPC) and pyruvate phosphate dikinase (PPDK) appeared to play a key role in ATP synthesis via substrate-level phosphorylation (SLP). Enzymes generating the antioxidant, reduced nicotinamide adenine dinucleotide phosphate (NADPH) were enhanced, while metabolic modules mediating the formation of the pro-oxidant, reduced nicotinamide adenine dinucleotide (NADH) were downregulated. CONCLUSIONS: Pseudomonas fluorescens reengineers its metabolic networks to generate ATP via SLP, a stratagem that allows the microbe to compensate for an ineffective electron transport chain provoked by excess Zn. SIGNIFICANCE AND IMPACT OF THE STUDY: The molecular insights described here are critical in devising strategies to bioremediate Zn-polluted environments.

Phosphate stress triggers the conversion of glycerol into l-carnitine in Pseudomonas fluorescens.

Glycerol, a by-product of the biofuel industry is transformed into l-carnitine when the soil microbe Pseudomonas fluorescens is cultured in a phosphate-limited mineral medium (LP). Although the biomass yield was similar to that recorded in phosphate-sufficient cultures (HP), the rate of growth was slower. Phosphate was completely consumed in the LP cultures while in the HP media, approximately 35 % of the initial phosphate was detected at stationary phase of growth. The enhanced production of α-ketoglutarate (KG) in HP cultures supplemented with manganese was recently reported (Alhasawi et al., 2017). l-carnitine appeared to be a prominent metabolite in the spent fluid while the soluble cellular-free extract was characterized with peaks attributable to lysine, γ-butyrobetaine (GB), acetate and succinate in the LP cultures. Upon incubation with glycerol and NH4Cl, the resting cells readily secreted l-carnitine and revealed the presence of such precursors like GB, lysine and methionine involved in the synthesis of this trimethylated moiety. Functional proteomic studies of select enzymes participating in tricarboxylic acid cycle (TCA), oxidative phosphorylation (OP), glyoxylate cycle and l-carnitine synthesis revealed a major metabolic reconfiguration evoked by phosphate stress. While isocitrate dehydrogenase-NAD+ dependent (ICDH-NAD+) and Complex I were markedly diminished, the activities of γ-butyrobetaine aldehyde dehydrogenase (GBADH) and l-carnitine dehydrogenase (CDH) were enhanced. Real-time quantitative polymerase chain reaction (RT-qPCR) analyses pointed to an increase in transcripts of the enzymes γ-butyrobetaine dioxygenase (bbox1), S-adenosylmethionine synthase (metK) and l-carnitine dehydrogenase (lcdH). The l-carnitine/γ-butyrobetaine antiporter (caiT) was enhanced more than 400-fold in the LP cultures compared to the HP controls. This metabolic reprogramming modulated by phosphate deprivation may provide an effective technology to transform glycerol, an industrial waste into valuable l-carnitine.

Aluminum-induced defective mitochondrial metabolism perturbs cytoskeletal dynamics in human astrocytoma cells.

Although aluminum (Al), a known environmental toxin, has been implicated in a variety of neurological disorders, the molecular mechanism responsible for these conditions is not fully understood. In this report, we demonstrate the ability of Al to trigger mitochondrial dysfunction and ineffective adenosine triphosphate (ATP) production. This situation severely affected cytoskeletal dynamics. Whereas the control cells had well-defined structures, the Al-exposed astrocytoma cells appeared as globular structures. Creatine kinase (CK) and profilin-2, two critical modulators of cellular morphology, were markedly diminished in the astrocytoma cells treated with Al. Antioxidants such as alpha-ketoglutarate and N-acetylcysteine mitigated the occurrence of the globular-shaped cells promoted by Al toxicity. Taken together, these data reveal an intricate link between ATP metabolism and astrocytic dysfunction and provide molecular insights into the pathogenesis of Al-induced neurological diseases.

Blue native polyacrylamide gel electrophoresis and the monitoring of malate- and oxaloacetate-producing enzymes.

We demonstrate a facile blue native polyacrylamide gel electrophoresis (BN-PAGE) technique to detect two malate-generating enzymes, namely fumarase (FUM), malate synthase (MS) and four oxaloacetate-forming enzymes, namely pyruvate carboxylase (PC), phosphoenolpyruvate carboxykinase (PEPCK), citrate lyase (CL) and aspartate aminotransferase (AST). Malate dehydrogenase (MDH) was utilized as a coupling enzyme to detect either malate or oxaloacetate in the presence of their respective substrates and cofactors. The latter four oxaloacetate-forming enzymes were identified by 2,6-dichloroindophenol (DCIP) and p-iodonitrotetrazolium (INT) while the former two malate-producing enzymes were visualized by INT and phenazine methosulfate (PMS) in the reaction mixtures, respectively. The band formed at the site of enzymatic activity was easily quantified, while Coomassie staining provided information on the protein concentration. Hence, the expression and the activity of these enzymes can be readily evaluated. A two-dimensional (2D) BN-PAGE or SDS-PAGE enabled the rapid purification of the enzyme of interest. This technique also provides a quick and inexpensive means of quantifying these enzymatic activities in normal and stressed biological systems.

Brain metabolism and Alzheimer's disease: the prospect of a metabolite-based therapy.

The brain is one of the most energy-demanding organs in the body. It has evolved intricate metabolic networks to fulfill this need and utilizes a variety of substrates to generate ATP, the universal energy currency. Any disruption in the supply of energy results in various abnormalities including Alzheimer's disease (AD), a condition with markedly diminished cognitive ability. Astrocytes are an important participant in maintaining the cerebral ATP budget. However, under oxidative stress induced by numerous factors including aluminum toxicity, the ability of astroctyes to generate ATP is impaired due to dysfunctional mitochondria. This leads to globular, glycolytic, lipogenic and ATP-deficient astrocytes, cerebral characteristics common in AD patients. The reversal of these perturbations by such natural metabolites as pyruvate, α-ketoglutarate, acetoacetate and L-carnitine provides valuable therapeutic cues against AD.

A novel role for calcite in calcium homeostasis.

Calcium carbonate (CaCO3) minerals are known to be deposited in a wide array of different organisms, ranging from microbes to vertebrates [(1989) On Biomineralization, Oxford University Press, New York]. Calcite, aragonite and vaterite are the major crystalline structural polymorphs of CaCO3 associated with living systems, and participate in a variety of biological functions [(1989) Biomineralization: Chemical and Biochemical Perspectives, VCH Publishers, Weinham, Germany; (1991) Advances in Inorganic Chemistry 36, 137-200]. Here we report on the ability of a soil bacterium to synthesize calcite in a calcium-stressed environment. The elaboration of this exocellular crystalline residue enables the organism to regulate its calcium content. The attainment of calcium homeostasis via the exocellular deposition of bacterial calcite with unique crystal habits is a novel biological phenomenon.

Aluminum detoxication mechanism in Pseudomonas fluorescens is dependent on iron.

The soil microbe Pseudomonas fluorescens has been shown to detoxify aluminum by the elaboration of a soluble metabolite where the trivalent metal is sequestered [Appanna and St. Pierre, FEMS Microbiol. Lett. 24 (1994) 327-332]. The inclusion of 5 mM iron in the growth medium elicited an entirely disparate detoxification strategy. In this instance, the two trivalent metals were immobilized in a gelatinous lipid-rich residue. Dialysis and ultracentrifugation studies indicated that the test metals were being transformed from early stages of growth and were associated with phosphatidylethanolamine. However, at 45 h of cellular multiplication, most of the metals were deposited as an insoluble residue. X-ray fluorescence analyses identified the constituents of this mineral essentially as aluminum, iron and phosphorus. Scanning electron microscopy and energy dispersive X-ray microanalysis of the dialysate, isolated at 35 h of microbial growth, revealed thread-like structures associated with nodule-like bodies that were rich in the two test metals. Transmission electron microscopic studies aided in the visualization of iron and aluminum inclusions within the bacterial cells.

Exocellular and intracellular accumulation of lead in Pseudomonas fluorescens ATCC 13525 is mediated by the phosphate content of the growth medium.

Pseudomonas fluorescens appears to elicit disparate lead detoxification mechanisms in phosphate-rich and phosphate-deficient media. When grown in the presence of 0.1 mM Pb2+ complexed to citrate, the sole source of carbon, only a slight diminution in cellular yield was observed in the former medium. However, in a phosphate-deficient milieu, lead imposed approximately a 30% reduction in bacterial multiplication. At stationary phase of growth, 72% of the metal was found in the bacterial cells from the phosphate-deficient medium, while that from phosphate-rich broth contained only 12.5%. The latter medium was characterized by an insoluble pellet that accounted for 73.5% of the lead. Although no citrate was detected in the phosphate-rich media after 40 h of incubation, only 72% of citrate was consumed even after 70 h of growth in the phosphate-deficient cultures. The inclusion of lead did not appear to enhance the production of either extracellular proteins or carbohydrates.

Gallium toxicity and adaptation in Pseudomonas fluorescens.

When cultured in a defined citrate medium supplemented with 1 mM gallium (III) Pseudomonas fluorescens ATCC 13525 experienced a lag phase of 40 h with no apparent diminution in cellular yield. Following initial uptake of the metal-ligand complex, gallium was secreted in the spent fluid. This lag phase was abolished either by inoculating the medium with gallium adapted cells or by inclusion of iron (III) (20 microM) in the growth medium. In the culture enriched with both gallium and iron (III), X-ray fluorescence spectra revealed a gradual decrease of gallium from the spent fluid as growth progressed. In a phosphate deficient medium, no cellular multiplication was observed in the presence of gallium. The inhibitory influence mediated by the trivalent metal was reversed by the addition of (20 microM) iron (III). Although bacterial growth was accompanied by an initial decrease in exocellular gallium, a marked increment in the concentration of this metal was observed in the spent fluid at stationary phase of growth. Citrate was not detected in the exocellular fluid at cessation of bacterial multiplication. Electrophoretic analyses revealed numerous variations in the cytoplasmic protein profiles of the control and metal stressed cells. Gallium induced the syntheses of polypeptides with apparent molecular masses of 89 kDa, 50 kDa, 39 kDa, 26 kDa and 12 kDa.

Cellular response to a multiple-metal stress in Pseudomonas fluorescens.

Pseudomonas fluorescens multiplied in a minimal mineral medium supplemented with millimolar amounts of aluminum (5 mM), iron (5 mM), zinc (3 mM), calcium (2 mM) and gallium (1 mM). A slight decrease in growth rate and a 22% diminution in cellular yield were observed as compared to the control medium. Citrate, the sole source of carbon to which the test metals were complexed, was completely utilized. Although at stationary phase of growth most of the metals were immobilized in an exocellular lipid-rich residue, ultracentrifugation and dialysis studies revealed that metals were associated with phosphatidylethanolamine (PE) from early stages of growth. As growth progressed the metal content of the soluble cellular extract increased reaching an optimum at 35 h of incubation. However, no detectable amounts of metals in this cellular component were discerned at stationary phase of growth. There appeared to be no marked variation in exocellular protein and carbohydrate production in control and metal-stressed cultures. Transmission electron microscopic studies revealed metal rich bodies associated with the cytoplasm. Scanning electron microscopic analyses of the dialyzate aided in the identification of the metal-rich bodies associated with elongated structures comprised of carbon, oxygen and phosphorus. PE appeared to be an important organic constituent of the gelatinous residue.

Modulation of TCA cycle enzymes and aluminum stress in Pseudomonas fluorescens.

Oxalic acid plays a pivotal role in the adaptation of the soil microbe Pseudomonas fluorescens to aluminum (Al) stress. Its production via the oxidation of glyoxylate necessitates a major reconfiguration of the enzymatic reactions involved in the tricarboxylic acid (TCA) cycle. The demand for glyoxylate, the precursor of oxalic acid appears to enhance the activity of isocitrate lyase (ICL). The activity of ICL, an enzyme that participates in the cleavage of isocitrate to glyoxylate and succinate incurred a 4-fold increase in the Al-stressed cells. However, the activity of isocitrate dehydrogenase, a competitor for the substrate isocitrate, appeared to be diminished in cells exposed to Al compared to the control cells. While the demand for oxalate in Al-stressed cells also negatively influenced the activity of the enzyme alpha-ketoglutarate dehydrogenase complex, no apparent change in the activity of malate synthase was recorded. Thus, it appears that the TCA cycle is tailored in order to generate the necessary precursor for oxalate synthesis as a consequence of Al-stress.

Oxalic acid production and aluminum tolerance in Pseudomonas fluorescens.

13C NMR studies on intact cells from Al-stressed Pseudomonas fluorescens incubated with citric acid or Al-citrate yielded peaks at 158 and 166 ppm that were attributable to free and complexed oxalic acid, respectively. The presence of oxalic acid was further confirmed with the aid of oxalate oxidase. These peaks were not discernable in experiments performed with cells taken from control cultures. Enzymatic analyses of cell fractions showed the highest production of oxalic acid in the inner membrane fraction of Al-stressed cells incubated with glyoxylate. There was an eight-fold increase in the synthesis of oxalic acid in the inner membrane fraction from the Al-stressed cells compared to the control cells. Although oxalic acid production was observed when citrate, Al-citrate and isocitrate were utilized as substrates, the inner membrane fraction did not mediate the formation of oxalic acid from glycine/pyruvate, glycolic acid, oxaloacetate or ascorbate. These data suggest that the increased oxalic acid production in response to Al stress is effected via the oxidation of glyoxylate.

Indium detoxification in Pseudomonas fluorescens.

The interaction between indium, a non-essential toxic element, and a soil bacterium was studied. Although the presence of 0.5 mm indium complexed to citrate, the sole source of carbon, had an inhibitory influence on growth rate and cellular yield, Pseudomonas fluorescens circumvented the toxicity of the trivalent metal via its insolubilization as a phosphorus residue. The inclusion of 20 microm iron (III) arrested the negative impact of indium and no diminution of cellular yield was recorded. In this instance indium homeostasis was also attained by elaboration of an extracellular phosphorus-containing deposit. Electrophoretic analyses of the cytoplasmic extracts revealed several dissimilar patterns. Notably, two polypeptides with apparent molecular masses of 57 kDa and 18 kDa were induced in the metal-stressed bacteria. An increment in extracellular carbohydrates in metal-supplemented media was observed. No citrate was detected in the spent fluid at the cessation of cellular bilization may have potential application in metal pollution management.

The overexpression of NADPH-producing enzymes counters the oxidative stress evoked by gallium, an iron mimetic.

Gallium (Ga), an iron (Fe) mimetic promoted an oxidative environment and elicited an antioxidative response in Pseudomonas fluorescens. Ga-stressed P. fluorescens was characterized by higher amounts of oxidized lipids and proteins compared to control cells. The oxidative environment provoked by Ga was nullified by increased synthesis of NADPH. The activity and expression glucose 6-phosphate dehydrogenase (G6PDH) and isocitrate dehydrogenase-NADP (ICDH) were stimulated in Ga-cultures. The induction of isoenzymes of these dehydrogenases was also evident in the Ga-stressed cells. Although superoxide dismutase (SOD) activity was significantly enhanced in Ga-stressed cultures, catalase activity experienced a marked diminution. Fe metabolism appeared to be severely impeded by Ga toxicity. This is the first demonstration of the oxidative stress evoked by Ga to be neutralized by a reductive environment generated via the overexpression of NADPH-producing enzymes.

Galactosyl residue in exopolysaccharide from Rhizobium meliloti JJ-1 exposed to manganese in furanoid.

Exopolysaccharide (EPS) elaborated by Rhizobium meliloti JJ-1 in a manganese supplemented medium was isolated. Periodate oxidation, reduction with sodium borohydride, followed by hydrolysis and subsequent capillary gas liquid chromatography of the derived alditol acetates revealed that D-galactose in this complex biopolymer is in furanoid form. This observation was further confirmed by 13carbon nuclear magnetic resonance (13C NMR).

Aluminum Elicits Exocellular Phosphatidylethanolamine Production in Pseudomonas fluorescens.

Pseudomonas fluorescens ATCC 13525 was found to grow in a minimal mineral medium supplemented with millimolar amounts of aluminum, a known environmental toxicant. During the stationary phase of growth, the trivalent metal was localized in a phosphatidylethanolamine (PE)-containing residue. The concentration of PE in pellets ranged from 1.7 to 13.9 mg ml of culture(sup-1) in media supplemented with 1 to 30 mM aluminum. Although the gelatinous residue was observed during the stationary phase of growth, ultracentrifugation and dialysis experiments revealed that PE was produced from earlier stages of incubation and was associated with aluminum. A sharp diminution in the levels of PE and aluminum in the spent fluid was concomitant with the formation of the insoluble deposit. The aluminum content of the soluble cellular fraction increased during growth and reached an optimum of 1.85 mM of test metal at 45 h in cultures with 15 mM aluminum. Further incubation, however, led to a marked decrease in the cellular aluminum content, and during the stationary phase of growth, only trace amounts of the trivalent metal were detected in this fraction. When 45-h cells were incubated in fresh citrate medium, most of the intracellular aluminum was secreted in the spent fluid and citrate was rapidly consumed. Aluminum efflux was also observed in cultures in which d-glucose was substituted for citrate. However, no efflux of this trivalent metal was evident in media devoid of either citrate or d-glucose. Scanning electron microscopic studies and X-ray energy-dispersive analyses of the dialyzed supernatant aided in the visualization of nodule-like aluminum- and phosphorus-rich bodies associated with thread-like carbon-, oxygen-, and phosphorus-containing structures. Transmission electron microscopic and electron energy loss spectroscopic analyses revealed the presence of aluminum within bacteria after 45 h of incubation. Cells harvested after aluminum insolubilization did not shown aluminum inclusions. This aluminum-tolerant microbe may have potential application in bioremediation processes.

Aluminium, chromium and manganese detoxification mechanisms in Pseudomonas syringae: an X-ray fluorescence study.

Pseudomonas syringae cultured in a defined citrate medium supplemented with 1 mM aluminium, chromium and manganese, respectively, appeared to elicit disparate biochemical responses. At the stationary phase of growth aluminium was predominantly present as an insoluble residue. Although virtually none of this metallic element was detected in the supernatant, the bacterial cells appeared to contain some aluminium. Following the initial uptake of chromium the microbe secreted the metal in the supernatant. Only a small fraction of the chromium was localised in the bacterial cells; 91% manganese was biotransformed into an insoluble pellet. No citrate was detected in the exocellular fluid at cessation of cellular growth.